Tuning Microscale Substrate Mechanics by Stereolithography to Control Mesenchymal Stromal Cell Functions

Cell volume regulation is an essential component to a myriad of functions—including migration, proliferation and communication. In a physiological environment, extracellular matrices (ECM) have complex intrinsic (e.g., elasticity) and extrinsic (e.g., confinement) mechanical properties; how microscale heterogeneity in these properties impacts volume control and downstream functions remains unclear. Here, we leveraged freeform stereolithography to fabricate microscale hydrogel posts to serve as discrete 3D matrix fibers for cell attachment. We built a custom print head and hydrogel chamber into a three-axis printer to deliver single, focused UV impulses that rapidly crosslink hydrogel microposts. By modulating laser exposure parameters (power and dwell time) at precise locations, we can specify the coordinates and elastic moduli of individual microposts—thereby fabricating scaffolds to vary the mechanics and confinement provided to individual cells. We find that the volume of human mesenchymal stromal cells (hMSCs) decreases in physiologically stiff (~20 kPa) scaffolds when they are more confined. In contrast, the volume of cells in soft scaffolds (~4 kPa) increases in confinement as cells deform soft posts to increase the number of post interactions. Strikingly, simultaneous presence of soft and stiff microposts enables confined hMSCs to deform more stiff microposts, leading to hyperallometric increases in cell volume through myosin-II dependent mechanisms and upregulation of monocyte regulatory genes upon inflammatory activation. The results underscore the cooperativity between soft and stiff subtrates with broad implications in mechanobiology of cell size regulation, immunomodulation and regenerative medicine.